In vitro and in vivo evaluation of clastogenicity of second-line antitubercular drug loaded PLGA nanoparticles.

Sustained release nanoformulations of second line antitubercular drugs levofloxacin and ethionamide had shown promise in pharmacokinetics and acute and sub-acute toxicity studies. The present study evaluated the clastogenicity potential of the nanoformulations of these antitubercular agents. Clastogenicity was evaluated by (a) in vitro micronucleus assay (b) in vivo micronucleus assay in Swiss albino mice and (c) sister chromatid exchange (SCE) in CHO cell lines. Ethionamide and levofloxacin loaded nanoparticles were 312 ± 64 nm and 245 ± 24 nm in size respectively and drug encapsulation was 35.2 ± 3.1% w/w and 45.6 ± 9.4% w/w, respectively. The frequency of MN-NCE/1000 NCE and MN-PCE/1000 PCE were significantly reduced in mice treated with ethionamide nanoparticle (3.5 ± 0.9, 13.8 ± 16.68) and levofloxacin nanoparticles (5.6 ± 2.7, 16.7 ± 12.7) compared to the mice treated with free ethionamide (11.5 ± 4.1, p = 0.23 and 45.19 ± 19.21, p = 0.38) and free levofloxacin (14.7 ± 1.88, p < 0.0001 and 54.6 ± 18.1, p = 0.0017), respectively. For in vitro, micronucleus assay frequencies of micronuclei per thousand bi-nucleated cells (MN-BN/1000 BN) was 188.3 ± 20.20 and 148 ± 20.42 for ethionamide and levofloxacin nanoparticles as compared to 232.6 ± 16.04 (p = 0.52) and 175 ± 5.56 (p = 0.45) for free ethionamide and levofloxacin, respectively. The average number of SCE per cell for nanoformulation of ethionamide were not different from that of free drug (4.9 ± 0.51 vs 4.1 ± 0.55, p = 0.86). The SCE per cells were not significant difference for nanoformulation of levofloxacin (2.33 ± 1.36 vs 5.46 ± 0.25, p = 0.88). In vitro and in vivo assays have shown relatively less clastogenic potential of equivalent dose of ethionamide nanoparticles as compared to the conventional formulation.

Risk Factors for Transfusion Transmissible Infections Elicited on Post Donation Counselling in Blood Donors: Need to Strengthen Pre-donation Counselling.

Donor notification and counselling transforms the legal and ethical requirement of disclosure of transfusion transmissible infection (TTI) in a blood donor into practice. The present study was done to assess the response to the disclosure of TTI reactivity results in blood donors, assess the risk factors in blood donors and follow the compliance of the disclosure and clinical referral in a population of blood donors who are difficult to convince that they may be harbouring infections apparently in a healthy state today but with possible clinical disease consequences in the future. A retrospective study was conducted from April 2011 to November 2012. Screening was done using third generation ELISA kits used according to the manufacturer's directions; these kits were approved for use in blood banks by the Drug Controller General of India. Those testing repeat reactive were referred for further confirmation and management. The total number of TTI reactive donors was 787 (0.93 %, N = 83,865). The observed response rate in the present study is 21.6 % (167, N = 787). The risk factors for acquiring infections in TTI reactive donors were statistically significant history of high risk behaviour (20.3 %) for human immunodeficiency virus infection and history of jaundice in themselves, family or close contacts (16.1 %) for hepatitis B virus infection. One hundred and ten (65.8 %) of the referred donors were on outpatient clinical care when post-referral follow up was conducted. The study emphasises on continuing sensitization of blood donation camp organisers to the need of privacy during blood donor selection. The study also stresses the need to strengthen the pre-donation counselling at outdoor blood donation at the same time raise awareness amongst blood donors about the importance of post-donation counselling and follow up.

Preproinsulin specific CD8+ T cells in subjects with latent autoimmune diabetes show lower frequency and different pathophysiological characteristics than those with type 1 diabetes.

Latent autoimmune diabetes in adults (LADA) resembles type 1 diabetes (T1D) in disease presentation except that its onset is slow. We compared pathophysiological characteristics of CD8+ T cells recognizing preproinsulin (PPI) derived epitopes in both disease groups using MHC-I dextramers (DMRs) in peripheral blood and after in-vitro stimulation with PPI. Subjects with T1D harbored higher frequency of DMR+ CD8+ T cells with relatively higher frequency of effector T cell subsets. Following stimulation with PPI, an increase in DMR+ CD8+ T cells, particularly the central-memory subset was observed in T1D group, whereas no significant change in DMR+ CD8+ T cell subsets was observed in LADA group. Intracellular expression of Granzyme-B and Perforin in DMR+ CD8+ T cells was comparable in both the groups. In conclusion, lower frequency and inferior proliferative potential on account of a relatively restrained central-memory subset of PPI specific CD8+ T cells are associated with slow rate of disease progression in LADA.

Paromomycin-loaded albumin microspheres: efficacy and stability studies.

In the present work, paromomycin-loaded albumin microspheres (PM-MS) have been formulated for passive targeting of paromomycin (PM) to macrophages, for the treatment of visceral leishmaniasis (VL). PM-MS were prepared by spray-drying method with a mean particle size of ≈ 3 µm. Thermal and chemical cross-linking methods were used for controlling drug release from the prepared microspheres (MS). PM-MS were then tested for efficacy and stability studies. In efficacy study, in vitro promastigote assay was carried out to assess the susceptibility of promastigote to PM in the concentration range of 5.0-150 µg/ml; cytotoxicity assay was performed to determine possible toxicity of PM for the host cells (peritoneal macrophages) and intracellular amastigote assay was carried out to determine the efficacy of free PM (PM solution) and encapsulated PM (PM-MS). Results obtained indicated a significant increase in efficacy of PM-MS in comparison to PM solution at equivalent concentration. Subsequently, stability studies of prepared formulation was carried out at various temperature and humidity conditions, these studies provided stability of formulation at all tested conditions including accelerated conditions. Thus, it can be concluded that present work provides an optimized formulation with stability and enhanced efficacy.

Terpenoidal constituents of Eucalyptus loxophleba ssp. lissophloia.

CONTEXT: Eucalyptus has been a source of a number of biologically active compounds. The anti-leishmanial activity of terpenoids from Eucalyptus loxophleba (Benth.) ssp. lissophloia (Myrtaceae) has not yet been investigated. OBJECTIVE: Isolation of the terpenoidal constituents for evaluation of in vitro anti-leishmanial activity against the Leishmania donovani (Dd8 strain) promastigotes. MATERIALS AND METHODS: The chloroform-methanol (8:2) extract of dried leaves of Eucalyptus loxophleba was used to isolate terpenoidal constituents employing solvent partitioning, column chromatography and preparative high performance liquid chromatography and characterized from spectral data. The anti-leishmanial activity of the isolated compounds was tested in vitro using an Alamar blue assay against a culture of L. donovani (Dd8 strain) promastigotes. RESULTS: Two new naturally occurring triterpenes, named loxanic acid and 3-acetyl loxanic acid together with four known ursane triterpenoids and one bis-monoterpene glycoside, cuniloside B isolated from the leaves showed anti-leishmanial activity (IC(50) 133 to 235 µM) against the promastigotes of the tested strain. CONCLUSION: The terpenes isolated from the leaves of E. loxophleba showed moderate anti-leishmanial activity.

Formylated phloroglucinols from Eucalyptus loxophleba foliage.

Two new naturally occurring formylated phloroglucinol compounds (FPCs), a dimer, loxophlebal B (10) and a cyclized FPC, loxophlebene (8) together with eight other formylated phloroglucinols (1-7 and 9) were isolated from the chloroform-methanol (8:2) extract of the leaves of Eucalyptus loxophleba ssp. lissophloia. The structures of new compounds were established by comprehensive spectral analysis and by comparison of their NMR data with those of related compounds in the literature. All the isolated compounds were evaluated for anti-leishmanial activity against promastigotes of Leishmania donovani.

Synthesis and antileishmanial activity of piperoyl-amino acid conjugates.

Based on reported antileishmanial activity of naturally occurring alkaloid piperine and amino acid esters, their conjugates were synthesized by the hydrolysis of piperine to piperic acid followed by reaction with amino acid methyl esters. These conjugates were further converted to compounds with free carboxyl group and those with reduced double bonds. The synthesized compounds were evaluated for activity against promastigote and amastigote forms of L. donovani in vitro. All the compounds showed better activity than either piperine or the amino acid methyl esters. Piperoyl-valine methyl ester was the most active compound showing an IC50 of 0.075 mM against the amastigotes. Two active compounds were evaluated for in vivo activity in golden hamster model of leishmaniasis.

Potential role of CXCL10 in the induction of cell injury and mitochondrial dysfunction.

Chemokines have been known to play a critical role in pathogenesis of chronic pancreatitis and acinar cell death. However, the role played by one of the CXC chemokines: CXCL10 in regulation of acinar cell death has remained unexplored. Hence, this study was designed to assess the role of CXCL10 promoting apoptosis in ex vivo cultured acinar cells. Primary human pancreatic acinar cell cultures were established and exposed to varying doses of CXCL10 for different time intervals. Apoptotic induction was evaluated by both qualitative as well as quantitative analyses. Various mediators of apoptosis were also studied by Western blotting, membrane potential (Psim) and ATP depletion in acinar cells. Analysis of apoptosis via DNA ladder and cell death detection - ELISA demonstrated that CXCL10 induced 3.9-fold apoptosis when administrated at an optimal dose of 0.1 mug of recombinant CXCL10 for 8 h. Quantitative analysis using FACS and dual staining by PI-annexin showed increased apoptosis (48.98 and 53.78% respectively). The involvement of upstream apoptotic regulators like pJNK, p38 and Bax was established on the basis of their increased expression of CXCL10. The change of Psim by 50% was observed in the presence of CXCL10 in treated acinar cells along with enhanced expression of Cytochrome C, apaf-1 and caspase 9/3 activation. In addition, ATP depletion was also noticed in CXCL10 stimulated acinar cells. CXCL10 induces cell death in human cultured pancreatic cells leading to apoptosis and DNA fragmentation via CXCR3 signalling. These signalling mechanisms may play an important role in parenchymal cell loss and injury in pancreatitis.

Role of anti-tubercular therapy in uveitis with latent/manifest tuberculosis.

PURPOSE: To assess the role of anti-tubercular therapy in uveitis with latent/manifest tuberculosis (TB). DESIGN: Retrospective, interventional case series. METHODS: A total of 360 patients from uveitis clinic with following inclusion criteria were studied: 1) complete clinical records of visual acuity, slit-lamp biomicroscopic examination, intraocular pressure, complications if any, and treatment records at the baseline and at all follow-up visits; 2) a documented positive tuberculin skin test (10 mm of induration or more) at 48 to 72 hours; 3) evidence of active uveitis, i.e., cellular reaction in the anterior chamber with or without keratic precipitates, and/or active vitreous inflammation, retinal vasculitis, choroiditis, or neuroretinitis; 4) all known causes of infectious uveitis except TB and known noninfectious uveitic syndromes ruled out; and 5) a minimum one year of follow-up from the initiation of treatment. Of these, 216 patients (Group A) received four-drug anti-tubercular therapy and corticosteroids, and 144 patients (Group B) received corticosteroids alone. The main outcome measure was recurrence of inflammation after minimum six months of initiating treatment in each group. RESULTS: Recurrences reduced significantly (P < .001) in Group A (15.74%) as compared to Group B (46.53%) over a median follow-up of 24 and 31 months, respectively. The patients treated with anti-tubercular therapy with corticosteroids had decreased risk of developing recurrence of uveitis by approximately two-thirds as compared to those treated with corticosteroids alone. CONCLUSION: Addition of anti-tubercular therapy to corticosteroids in uveitis patients with latent/manifest TB led to significant reduction in recurrences of uveitis.

Mitochondrial dysfunction and apoptosis of acinar cells in chronic pancreatitis.

BACKGROUND: The mechanism of acinar cell death in human chronic pancreatitis (CP) remains largely unexplored. Previous studies have demonstrated the role played by apoptosis and necrosis in experimental pancreatitis; however, their relationship with the progression of CP remains unknown. The present study was carried out to elucidate the role and extent of apoptosis in CP tissues with different histopathological scores and to examine the possible apoptotic pathway involved. METHODS: Pancreatic tissues (25 CP patients) that had been histopathologically graded (I-III) and ten normal pancreatic tissue samples were evaluated for apoptosis by DNA fragmentation and an in situ TUNEL assay. The expression of various apoptotic and antiapoptotic markers in the tissues were studied by immunohistochemistry and Western blotting. To elucidate the role of the mitochondria in acinar cell death, the mitochondrial membrane potential (DeltaPsim) and ATP levels were determined by flow cytometry and a luminometer. RESULTS: The presence of DNA fragmentation and apoptotic nuclei in all CP tissues confirmed the presence of apoptosis. The apoptotic index in CP tissue ranged from 0.09% to 0.86% +/- 0.02% and was highest in grade II (0.7 +/- 0.04%) tissues. Differential upregulation of the apoptotic mediators p53, Bax, cytochrome c, and caspase-3 and -9, and downregulation of antiapoptotic Bcl-2, was observed in CP. DeltaPsim on the order of 1.2-to 2.2-fold and ATP depletion in the range of 23%-84% in CP tissues was observed. CONCLUSIONS: Apoptosis plays an important role both in the initial stages and during the progression of CP, as evident in all tissue grades. Increased DeltaPsim, loss of ATP, and activation of caspases suggests the involvement of intrinsic pathways.

Primary culture of pancreatic (human) acinar cells.

The acinar cell culture plays a very important role in research of pancreatic pathophysiology. The aim of this study was to establish a long-term culture of human (foetal) pancreatic acinar cells in standardized nutrient media with supplements. Acinar cells were prepared from pancreatic tissues obtained from aborted foetus (> or =35 weeks) with no prior pancreatic complications by collagenase digestion and cultured using different media and supplements. The purity and phenotype of acinar cells was confirmed by various staining techniques and FACS. The acinar cell proliferation was determined at different time intervals by Bromo-deoxyuridine (BrdU) incorporation, and metabolic enzyme activity was analysed. The acini could be cultured and maintained in Ham's F-12 K/M199 media in the presence of 5% BSA, 0.1 mg/ml STI, 10 ng/ml EGF, and 10% FCS with the same morphological appearance as that of freshly prepared for 12 days with maximum viability of 80-85% and formation of monolayer without extracellular matrix. A significant BrdU incorporation of acinar cells in primary culture was observed which was maximum (105%) at day four. Higher amylase and lipase activity was seen in freshly isolated acinar cells which decreased with time of the culture. The established human pancreatic acinar cell culture may act as an excellent model to study exocrine dysfunction or pancreatitis in response to acinar cell injury.